Spermatogenesis is the process of germ cell proliferation and differentiation within the seminiferous tubules of the testis which leads to a haploid, free-swimming spermatozoon. Orchestrated in large measure by Sertoli cells, spermatogenesis requires complex endocrine and auto/paracrine regulation as well as direct cell-to-cell interaction (Parvinen et al., “Cell Interactions During the Seminiferous Epithelial Cycle,” Int. Rev. Cytol. 104:115-151 (1986); Kierszenbaum, “Mammalian Spermatogenesis In vivo and In vitro: A Partnership of Spermatogenic and Somatic Cell Lineages,” Endocr. Rev. 15:116-134 (1994); Griswold, “The Central Role of Sertoli Cells in Spermatogenesis,” Semin. Cell Dev. Biol. 9:411-416 (1998)). However, precise molecular mechanisms regulating the extensive cross-talk among various somatic and germ cell types remain to be established.
An effective procedure for recapitulating spermatogenesis in vitro would greatly facilitate mechanistic studies of the in vivo process while providing a biological basis for treating selected causes of male infertility and genetically modifying the male germ line. In vitro spermatogenesis (IVS) could be applied to generate developmentally competent haploid spermatids or sperm which could then be used in conjunction with round spermatid injection (ROSI) or intracytoplasmic sperm injection (ICSI) procedures (Palermo et al., “Pregnancies After Intracytoplasmic Sperm Injection of Single Spermatozoon into an Oocyte,” Lancet 340:17-18 (1992); Van Steirteghem et al., “Use of Assisted Fertilization,” Hum. Reprod. 8:1784-1785 (1993)) to overcome male factor infertility, especially azoospermia due to maturation arrest (Tesarik et al., “Restoration of Fertility by In-vitro Spermatogenesis,” Lancet 353: 555-556 (1999)). Also, transfection of diploid germ cells in culture followed by IVS would provide a direct approach to genetic modification of the male germ line (Brinster et al., “Spermatogonial Stem Cell Transplantation, Cryopreservation, and Culture,” Semin. Cell Dev. Biol. 9: 401-409 (1998)), including targeted gene insertion via homologous recombination.
Devising an IVS culture system that will support germ cell development through meiosis has been especially challenging. Miura et al. reported an organ culture system in which fragments of immature Japanese eel testis, containing only spermatogonia and inactive testicular somatic cells, were maintained and completed spermatogenesis in a chemically defined medium (Miura et al., “Hormonal Induction of All Stages of Spermatogenesis In vitro in the Male Japanese Eel (Anguilla japonica),” Proc. Natl. Acad. Sci. USA 88: 5774-5778 (1991)). In rodents, some stage-specific progression of spermatogenesis has been achieved in vitro utilizing organ culture or co-culture with immortalized Sertoli cells (Rassoulzadegan et al., “Transmeiotic Differentiation of Male Germ Cells in Culture,” Cell 75: 997-1006 (1993); Tajima et al., “Insulin-Like Growth Factor-I and Transforming Growth Factor-α Stimulate Differentiation of Type A spermatogonia in Organ Culture of Adult Mouse Cryptorchid Testes,” Int. J. Androl. 18:8-12 (1995); and Weiss et al., “Pre- and Postmeiotic Expression of Male Germ Cell-Specific Genes Throughout 2-Week Cocultures of Rat Germinal and Sertoli Cells,” Biol. Reprod. 57: 68-76 (1997)).
Mammalian germ cells can be maintained in culture for months during which they retain their full spermatogenic potential (Brinster et al., “Spermatogonial Stem Cell Transplantation, Cryopreservation, and Culture,” Semin. Cell Dev. Biol. 9: 401-409 (1998)), but difficulties in establishing conditions for germ cells to proceed to and through meiosis have limited the success of IVS culture systems (Kierszenbaum, “Mammalian Spermatogenesis In vivo and In vitro: A Partnership of Spermatogenic and Somatic Cell Lineages,” Endocr. Rev. 15:116-134 (1994)). Recently, however, meiosis of rat germ cells in culture has been reported using seminiferous tubule segments (Weiss et al., “Pre- and Postmeiotic Expression of Male Germ Cell-Specific Genes Throughout 2-Week Cocultures of Rat Germinal and Sertoli Cells,” Biol. Reprod. 57: 68-76 (1997); and Hue et al., “Meiotic Differentiation of Germinal Cells in Three-Week Cultures of Whole Cell Population from Rat Seminiferous Tubules,” Biol. Reprod. 59:379-387 (1998)). By measuring the expression of stage-specific markers phosphoprotein p19 and testis-specific histone TH2B (pachytene spermatocytes) and transition proteins T1 and T2 (round spermatids) as a function of days in culture, Hue et al. demonstrated an increase in the round spermatid to spermatocyte ratio during a 3-week culture (Hue et al., “Meiotic Differentiation of Germinal Cells in Three-Week Cultures of Whole Cell Population from Rat Seminiferous Tubules,” Biol. Reprod. 59:379-387 (1998)). The increased ratio corresponded to an increase in haploid cells in culture based on ploidy analysis. Retention of germ cell-Sertoli cell associations during tissue dissociation was considered to be a critical feature of this culture system. Tesarik et al. recently reported a live human birth following ROSI using spermatids recovered after in vitro differentiation of primary spermatocytes (Tesarik et al., “Restoration of Fertility by In-vitro Spermatogenesis,” Lancet 353: 555-556 (1999)). However, complete IVS from gonocytes through spermatocytogenesis, meiosis, and spermiogenesis has not been reported previously for mammalian species.
The complete recapitulation of spermatogenesis in vitro remains an elusive goal in reproductive biology. While germ cell viability and functionality can be maintained for extended periods in culture (Brinster et al., “Spermatogonial Stem Cell Transplantation, Cryopreservation, and Culture,” Semin. Cell Dev. Biol. 9: 401-409 (1998)), only limited differentiation of spermatogenic cells has been achieved in vitro (Kierszenbaum, “Mammalian Spermatogenesis In vivo and In vitro: A Partnership of Spermatogenic and Somatic Cell Lineages,” Endocr. Rev. 15:116-134 (1994)). Several reports in recent years have demonstrated that germ cells cultured in association with Sertoli cells can progress through discrete steps in the sequence of spermatogenesis during short-term culture, including initiation or completion of meiosis and initiation of spermiogenesis (Rassoulzadegan et al., “Transmeiotic Differentiation of Male Germ Cells in Culture,” Cell 75: 997-1006 (1993); Hue et al., “Meiotic Differentiation of Germinal Cells in Three-Week Cultures of Whole Cell Population from Rat Seminiferous Tubules,” Biol. Reprod. 59:379-387 (1998); Hofmann et al., “Immortalized Germ Cells Undergo Meiosis In vitro,” Proc. Natl. Acad. Sci. USA 91:5533-5537 (1994); and Tesarik et al., “In-vitro Differentiation of Germ Cells from Frozen Testicular Biopsy Specimens,” Hum. Reprod. 15:1713-1716 (2000)). However, long-term culture conditions that result in the progression of undifferentiated mammalian gonocytes through spermatocytogenesis and meiosis have not been reported.
The complex Sertoli-germ cell interactions required for the orchestration of spermatogenesis in vivo (Griswold, “The Central Role of Sertoli Cells in Spermatogenesis,” Semin. Cell Dev. Biol. 9:411-416 (1998)), including intercellular junctions and endocrine and paracrine regulation, appear to be critical to the in vitro process also, as culture systems utilizing tubule fragments (Hue et al., “Meiotic Differentiation of Germinal Cells in Three-Week Cultures of Whole Cell Population from Rat Seminiferous Tubules,” Biol. Reprod. 59:379-387 (1998)) or homogenates (Tesarik et al., “In-vitro Differentiation of Germ Cells from Frozen Testicular Biopsy Specimens,” Hum. Reprod. 15:1713-1716 (2000)) stimulate germ cell differentiation more successfully than other approaches. A potential limitation to this approach is the permeability barrier to critical medium constituents created by peritubular cells and basement membrane components that persist in tubule fragments. To avoid this potential constraint, peritubular cells can be removed and seminiferous tubules were completely dissociated enzymatically (Karl et al., “Sertoli Cells of the Testis: Preparation of Cell Cultures and Effects of Retinoids,” Methods Enzymol. 190:71-75 (1990)). Direct association among testis cells (germ cells and Sertoli cells) can be reestablished by aggregation with lectins such as concanavalin A (Grootegoed et al., “Concanavalin A-Induced Attachment of Spermatogenic Cells to Sertoli Cells In vitro,” Exp. Cell Res. 139:472-475 (1982)).
The present invention is directed to overcoming these and other deficiencies in the art.